1
Amer2 interacts with EB1 and APC and controls microtubule stability and cell migration*
Astrid S. Pfister1, Michel V. Hadjihannas
1, Waldemar Röhrig
1, Alexandra Schambony
2 and
Jürgen Behrens1
From the 1 Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nuremberg, Glückstr.
6, 91054 Erlangen, Germany
and the 2 Biology Department, Developmental Biology, University Erlangen-Nuremberg, Staudtstr. 5,
91058 Erlangen, Germany
Running title: Amer2 and EB1 control microtubule stability *
To whom correspondence should be adressed: Jürgen Behrens, Nikolaus-Fiebiger-Center, University
Erlangen-Nuremberg, Glückstr. 6, 91054 Erlangen, Germany. Fax: 0049-9131-8529111; E-mail:
Keywords: Amer2; APC; cell migration; EB1; microtubule stabilization.
Background: Amer2 localizes to the plasma
membrane, interacts with APC and regulates
Wnt signaling.
Results: Amer2 recruits the microtubule-
associated protein EB1 to the plasma membrane
and affects the stabilization of microtubules and
cell migration.
Conclusion: Amer2 is a novel regulator of
microtubule stability by interacting with EB1.
Significance: A novel membrane-associated
regulator of microtubule stabilization at the
plasma membrane was identified and shown to
affect cell migration.
SUMMARY
EB1 is key factor in the organization of
the microtubule cytoskeleton by binding to
the plus ends of microtubules and serving as a
platform for a number of interacting proteins
(+TIPs) that control microtubule dynamics.
Together with its direct binding partner
adenomatous polyposis coli (APC) EB1 can
stabilize microtubules. Here we show that
Amer2 (APC membrane recruitment 2), a
previously identified membrane associated
APC binding protein is a direct interaction
partner of EB1 and acts as regulator of
microtubule stability together with EB1.
Amer2 binds to EB1 via specific S/TxIP
motifs and recruits it to the plasma
membrane. Coexpression of Amer2 and EB1
generates stabilized microtubules at the
plasma membrane whereas knockdown of
Amer2 leads to destabilization of
microtubules. Knockdown of Amer2, APC, or
EB1 reduces cell migration, and morpholino-
mediated downregulation of Xenopus Amer2
blocks convergent extension cell movements
suggesting that the Amer2/EB1/APC complex
regulates cell migration by altering
microtubule stability.
INTRODUCTION The EB1 (end binding 1) protein was
initially identified as an interaction partner of the
C-terminal end of the tumour suppressor protein
APC (1). It was then shown to bind
preferentially at the plus ends of growing
microtubules and to dissociate rapidly from the
more mature microtubule lattice thereby
generating comet like structures that can be
visualized by fluorescence microscopy (2). EB1
recruits a variety of proteins to the microtubule
plus ends that control microtubule dynamics
suggesting that it represents a platform for
microtubule regulators. Because of their
association with growing microtubule ends, EB1
and its binding partners are collectively termed
microtubule plus end tracking proteins or +TIPs.
It was recently shown that +TIP proteins
associate with EB1 by short sequence stretches
containing S/TxIP amino acid motifs (3). In vitro
and in vivo studies have revealed in part
opposing effects of EB1 on different parameters
of microtubule dynamics, including
polymerisation, catastrophe frequency, pausing
and rescue (4). In mammalian cells and Xenopus
egg extracts, EB1 promotes microtubule growth
and stability, at least in part by lowering
catastrophe frequencies (5,6). APC is a +TIP
http://www.jbc.org/cgi/doi/10.1074/jbc.M112.385393The latest version is at JBC Papers in Press. Published on August 16, 2012 as Manuscript M112.385393
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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protein that can bind and stabilize microtubules
in clusters at the cell cortex (7,8). APC
cooperates with EB1 in the stabilization of
microtubules both in vitro and in vivo (9,10) but
may also localise and act independent of EB1 at
microtubules (7,11,12).
Besides its role in microtubule biology APC
has a well established function as a negative
regulator of the Wnt/β-catenin pathway by
promoting degradation of β-catenin (13). Among
other interaction partners it can bind to members
of the Amer protein family, consisting of
Amer1/WTX, Amer2 and Amer3 which share
conserved domains that interact with the N-
terminal armadillo repeats of APC (14,15).
Amer1 is a tumor suppressor and negative
regulator of Wnt signaling (15-17). Amer2 is a
membrane associated PtdIns(4,5)P2 binding
protein that interacts with APC via two
conserved APC binding domains and recruits it
to the plasma membrane (15,18). Amer2
negatively regulates Wnt signaling probably by
interfering with β-catenin (18). Here we show
for the first time that Amer2 directly interacts
with the microtubule associated protein EB1 and
recruits it to the plasma membrane. Moreover,
we reveal a role for Amer2 in regulating
microtubule stability presumably by providing a
platform for the microtubule binding proteins
APC and EB1 to promote cell migration.
EXPERIMENTAL PROCEDURES DNA constructs and siRNAs. The following
constructs have been described previously:
pcDNA-Flag-Amer2, pcDNA-Flag-Amer1 (15),
EB1-GFP (19), CMV-APC (20), pcDNA3.1-
Flag (21). Amer2-SKNN, TKNN and
SKNN/TKNN were generated by PCR
mutagenesis exchanging the amino acids IP to
NN. For expression of the GST-Amer2(559-671)
protein the cDNA encoding amino acids 559-671
of human Amer2 was amplified by PCR and
inserted into pGEX-4T3 (Amersham Pharmacia
Biotech). The sequences of siGFP and siAmer2
(termed siAmer2-1) have been described before
(18). siLuc: 5´-
CUUACGCUGAGUACUUCGA-3´; siEB1: 5´-
UUGCCUUGAAGAAAGUGAA-3´ (22);
siAPC: 5`-AAGACGUUGCGAGAAGUU-
GGA-3`. All siRNAs were purchased from
Dharmacon.
Antibodies. The rabbit Amer2 polyclonal
antibody was produced by immunising rabbits
with a recombinant GST-Amer2 fusion protein
containing amino acids 559-671 of human
Amer2 (Pineda, Berlin, Germany). The serum
was affinity purified using CNBr-activated
Sepharose 4B beads (GE healthcare) coupled to
the antigen. Commercial antibodies were
purchased from Sigma (rabbit anti-Flag; mouse
anti-Flag; mouse anti-acetylated Tubulin, clone
6-11B-1; rabbit anti-Pan Cadherin), Roche
(mouse anti-GFP, mixture of clones 7.1 and
13.1), Epitomics (rabbit anti-GFP),
BDTransduction Lab (mouse anti-EB1 clone 5),
Serotec (rat anti-α-Tubulin, clone YL1/2),
Abcam (mouse anti-APC, Ali (12-28)) and Cell
Signaling (rabbit anti-GAPDH, clone 14C10).
Secondary antibodies (Jackson
ImmunoResearch, Camebridgeshire, UK) were
either Cy2 and Cy3 conjugates for
immunofluorescence or HRP conjugates for
Western blotting.
Yeast two hybrid screen. Yeast two hybrid
and β-galactosidase assays were performed in
the L40 yeast strain using pBTM116 as a bait
vector and a mouse embryonic day 10.5 library
in pVP16 as described previously (21).
Cell culture and transfections. Cells were
cultured in 10% CO2 at 37°C in DMEM
supplemented with 10% FCS and 1%
penicillin/streptomycin (PAA laboratories,
Austria). Plasmid transfections were performed
using polyethylenimin for HEK293T cells,
TransIT-TKO reagent (Mirus, Madison, WI,
USA) for MCF-7 cells, and Lipofectamine2000
(Invitrogen) for U2OS cells. siRNAs were
transfected using Oligofectamine (Invitrogen)
for 48-72 h according to the manufacturer´s
instructions.
Preparation of protein lysates, subcellular
fractionation, co-immunoprecipitation and
Western blotting. Cells were washed with PBS
and lysed in Triton-X-100 buffer (20mM Tris-
HCl pH 7.4, 150mM NaCl, 5mM EDTA, 1%
Triton-X-100, 1mM DTT and 1mM PMSF) at
4°C for 10 minutes. Lysates were cleared at
13,000 rpm for 10 minutes at 4°C. For co-
immunoprecipitation, lysates were incubated
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over night with Anti-FLAG M2 affinity gel
beads (Sigma) or mouse GFP antibodies plus
protein A/G-Sepharose beads (Santa Cruz
Biotechnology). Immunoprecipitates were
collected, washed four times with NET low salt
buffer (50mM Tris-HCl, pH 8, 150mM NaCl,
5mM EDTA, 1% Triton-X-100) and eluted with
SDS sample buffer. Subcellular fractionation of
cells was carried out using the ProteoJET
Membrane Protein Extraction Kit (Fermentas)
according to the manufacturer´s instructions.
Western blotting was performed as reported (23).
Proteins were visualised using Enhanced
chemiluminescent reagent (Perkin Elmer) and a
luminoimager (LAS-3000, Fuji).
RT-PCR analysis was performed as reported
(18). The Amer2 and GAPDH primers for RT-
PCR have been described previously (18). The
sequences of the APC primers are: 5´-
AAGTTGCGGCCGCTGGG-
AACCAAGGTGGAAATGGTG-3´ and 5´-
AAGTCGCGGCCGCCTATTCAACAGGAGC
TGGCATTG.
Immunofluorescence stainings and
microscopy. For immunofluorescence staining
cells were grown on glass cover slips, fixed with
ice-cold methanol, permeabilised with 0.5%
Triton-X-100, blocked with DMEM/FCS and
stained with the indicated antibodies. To disrupt
microtubules, transiently transfected cells were
treated with nocodazole (2 µg/ml) for 1 h in the
incubator (10% CO2 at 37°C). Photographs were
taken with a CCD camera (Visitron, Munich,
Germany) on a Zeiss Axioplan 2 microscope
(Zeiss, Oberkochen, Germany) and MetaMorph
software (Molecular Devices). Images were
processed using Adobe photoshop CS software.
Cell Migration Assay. U2OS cells on
coverslips in 6 wells plates were transfected with
siRNAs and allowed to reach confluency. Three
wounds of defined size (approx. 850µm) were
made for each coverslip and cells were allowed
to migrate for 12 hours. Methanol fixed cells
were processed for α-tubulin
immunofluorescence staining. Measurements at
two positions along each of the three wounds
were taken and percentage closure as well as
statistical analysis (unpaired Student´s t-test)
were calculated.
Xenopus experiments. Embryos were
injected in both dorsal blastomeres at the 4-cell-
stage with 100 pg LacZ DNA plus 0.8 pmol of
Amer2-MO or control-MO as described
previously (18). At stage 12.5 embryos were
stained for LacZ and probed by in situ
hybridization for expression of XPAPC (24).
RESULTS Amer2 interacts with EB1 via S/TxIP motifs.
In a yeast two hybrid screen using a C-
terminal fragment of Amer2 as a bait we isolated
several interacting clones covering the C-
terminal part of EB1 (3) (Fig. 1A,B). The
Amer2-EB1 interaction was confirmed by co-
immunoprecipitation of the transiently expressed
full length proteins in HEK293T cells (Fig. 1C).
Endogenous EB1 was also co-
immunoprecipitated with transfected Amer2
(Fig. 1D). As a control, EB1 only weakly co-
immunoprecipitated with the related
Amer1/WTX protein (cf. Fig. 3A).
Amer2 is linked to the plasma membrane
through its interaction with phosphatidylinositol
phosphate lipids (18). Accordingly, Amer2
exhibited membrane and cytoplasmic staining
when expressed in MCF7 cells. Amer2 staining
was most prominent at the cell-cell contact areas
but was also occasionally observed at the
periphery of cells where there was no contact to
neighbouring cells. EB1 decorated microtubules,
as reported previously (Fig. 1E; (2,15)).
Importantly, when both proteins were
coexpressed, a large fraction of EB1 became
recruited to the plasma membrane colocalising
with Amer2 (Fig. 1E). As a control,
Amer1/WTX did not recruit EB1 to the plasma
membrane (Fig. 1E). Of note, a minor fraction of
Amer2 and EB1 was present at filamentous
structures possibly representing microtubules
which would point to recruitment of Amer2 to
microtubules by EB1 (Fig. 1F). Transiently
expressed Amer2 also recruited endogenous EB1
from microtubule comets, to the plasma
membrane (Fig. 1G). These data show that
Amer2 is a novel interaction partner of EB1 able
to recruit EB1 to the plasma membrane.
Various known +TIP proteins share a
specific four amino acid motif, S/TxIP which
makes direct contact to the C-terminal part of
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EB1 (3). We found two perfect matches of the
S/TxIP motif in our Amer2 bait, SKIP at amino
acid 606 and TKIP at amino acid 637 which are
conserved in Amer2 from different species (Fig.
2A). SKIP and TKIP motifs of Amer2 were
mutated to SKNN and TKNN, respectively, and
mutants were analyzed for EB1 interaction. In
yeast two hybrid assays mutations of either motif
alone did not affect interaction with the EB1
preys; however, mutation of both motifs
completely abrogated the interaction (Fig. 2B).
In co-immunoprecipitation experiments, which
provide more stringent conditions for
interactions, mutation of SKIP alone already
strongly reduced the binding of full length
Amer2 to EB1 whereas mutation of TKIP had a
minor effect. Binding to EB1 was completely
abolished in the double mutant Amer2-
SKNN/TKNN (Fig. 2C). In line, plasma
membrane recruitment of endogenous EB1 by
Amer2 was reduced but not completely
abolished by the SKIP mutation, whereas
mutation of TKIP had only a minor effect.
Recruitment of EB1 was completely abrogated
in the double mutant (Fig. 2D). These data show
that Amer2 binds directly to EB1 via the S/TxIP
consensus motifs, and that two of these motifs
are functional, albeit with different affinities
towards EB1.
Amer2 forms a scaffold for APC and EB1
complex formation.
APC interacts with Amer2 via its N-terminal
armadillo domain (15,18) and with EB1 via the
C-terminal EB1 binding domain (1). It might
therefore link EB1 to Amer2. Indeed, the amount
of EB1 co-immunoprecipitated with Amer2 was
greatly increased when APC was coexpressed
(Fig. 3A). Our data suggest that Amer2 acts as a
scaffold for a multiprotein complex containing
the microtubule interacting proteins EB1 and
APC (Fig. 3B).
Amer2 stabilizes microtubules together with
EB1.
Next we analyzed whether microtubule stability
is affected by Amer2 using the occurrence of
acetylated tubulin as a marker for stable
microtubules (25). In MCF cells, coexpression of
Amer2 and EB1 led to a marked increase and
concomitant enrichment of stabilized
microtubules at the cell cortex close to the
plasma membrane, in line with the preferential
membrane localization of Amer2 and EB1 in
these cells (Fig. 4Aa,b). In contrast,
coexpression of EB1 with the Amer2-
SKNN/TKNN mutant (Fig. 4Ac,d) or
transfection of Amer2 alone (Fig. 4Ae,f) did not
significantly alter intensity and distribution of
acetylated microtubules, indicating that direct
interaction of both proteins is required for
microtubule stabilization. Coexpression of
Amer2 and EB1 did not alter the general pattern
of the microtubule network as revealed by
staining with an antibody to α-tubulin (data not
shown).
To analyse whether Amer2 is required for
microtubule stability we performed loss of
function experiments. Indeed, knockdown of
Amer2 in HEK293T and HeLa cells led to a
marked decrease of acetylated tubulin as
determined by Western blotting (Fig. 4B). In
line, immunofluorescence staining revealed that
Amer2 knockdown strongly reduced the number
of stable microtubules in HeLa cells (Fig. 4C).
Acetylated tubulin levels were also reduced after
knockdown of APC or EB1 (Fig. 4D). To
address the point of microtubule stabilization by
Amer2/EB1 in a different experimental setup,
Amer2/EB1 cotransfectants of U2OS cells were
treated with low doses of nocodazole in order to
disrupt microtubules and then stained with anti-
α-tubulin antibodies. This revealed focal
retention of microtubules at areas of EB1
membrane localization suggesting that
microtubules associated with Amer2/EB1 are
resistant against nocodazole treatment. In
contrast, no such stabilization was observed in
cells expressing EB1 together with the Amer2-
SKNN/TKNN mutants deficient for EB1 binding
(Fig. 4E). Together our data suggest that Amer2
stabilizes microtubules in conjunction with EB1
and APC.
Amer2, EB1 and APC are required for
directed cell migration.
Microtubules are required for cell migration by
providing a basis for cell polarity (26).
Knockdown of Amer2 in U2OS cells clearly
abrogated cell migration as determined by
wounding assays (Fig. 5A). Knockdown of
either EB1 or APC also reduced cell migration
confirming previous publications (10,27) and
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suggesting similar roles of Amer2, EB1 and
APC in cell migration. To confirm a role of
Amer2 in cell migration in vivo, we knocked
down its expression in Xenopus using
morpholino oligonucleotides (18). We observed
defects in convergent extension movements
indicated by reduction of the XPAPC expressing
paraxial mesoderm as well as increased length
and width of the negatively stained notochord in
XAmer2 depleted embryos (Fig. 5B).
DISCUSSION In this study we have identified Amer2 as a
novel key factor in the control of the microtubule
cytoskeleton. Amer2 binds to APC and EB1 via
distinct binding domains and seems to cooperate
with EB1 in microtubule stabilization. Whereas
expression of Amer2 and EB1 alone had no or
only minor effects on the stability of
microtubules, coexpression of both proteins
generated stable bundles of microtubules at the
cell cortex close to the plasma membrane.
Conversely, reduction in Amer2 levels similar to
reduction of EB1 and APC strongly reduces the
number of stabilized microtubules. Of note APC
increases the amounts of EB1 associated with
Amer2 by linking it to the complex (Fig. 3). We
propose a model in which Amer2 acts as a
scaffold for EB1 and APC to coordinate their
functional interaction with microtubules. Both
EB1 and APC were shown to be able to stabilize
microtubules by either acting separately or
together (9,10). It was suggested that
endogenous APC and EB1 colocalize only
transiently at microtubule tips (7,11). Bridging
of both factors by Amer2 might foster their
cooperation in microtubule stabilization, for
instance by increasing the local concentration of
these factors at microtubule ends. Interestingly,
a similar triple complex of EB1 and APC with
the Rho GTPase effector mDia was shown to
stabilize microtubules downstream of Rho
signaling (10).
Based on its specific interaction with EB1 via
S/TXIP motifs Amer2 resembles classical +TIP
proteins. Similar to these, Amer2 might track
microtubule plus ends via its association with
EB1, e.g. during transport to the plasma
membrane. However, we have mainly seen
Amer2 at the plasma membrane, sometimes also
at fibres together with EB1 (Fig. 1F), but never
detected it at microtubule ends suggesting that
the Amer2/EB1/APC complex forms
predominantly at the plasma membrane and not
at growing microtubules. Membrane association
of Amer2 is mediated by PtdIns(4,5)P2
suggesting that occurrence of this lipid
determines the differential localization of
Amer2/EB1 (18). Wnt signaling was shown to
stimulate synthesis of PtdIns(4,5)P2 and we
could recently show that the related Amer1
becomes membrane associated in a
PtdIns(4,5)P2-dependent manner after
stimulation with Wnt3A (28). In similar
experiments we noticed that Amer2 shifted to the
plasma membrane fraction after Wnt3A
treatment (data not shown). Thus, Wnt signaling
might induce membrane association of Amer2
which in turn might recruit EB1 and APC. The
consequences of such a mechanism for cortical
association of microtubules needs to be further
explored. Amer2 resembles the membrane
associated LL5β protein that interacts with
CLASP +TIPs and is required for cortical
attachment of microtubules. Similar to Amer2
LL5β requires binding to phospholipids, in
specific PIP3 for membrane recruitment, and PI3
kinase signaling was suggested to regulate
membrane association of LL5β (29).
Microtubule organization is a prerequisite for
directed cell migration, probably because it
imposes cell polarity on the migrating cells (26).
In line, microtubules are more stable at the
leading than at the trailing edge of migrating
cells (30). Our knockdown experiments show
that Amer2, APC and EB1 are similarly required
for cell migration, probably due to their effects
on microtubule stability. Moreover, morpholino-
mediated downregulation of XAmer2 resulted in
defects in convergent-extension cell movements
during gastrulation. Thus, Amer2 appears to be a
hub for cellular activities linking the
microtubule-based cytoskeleton to cell
migration.
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29. Lansbergen, G., Grigoriev, I., Mimori-Kiyosue, Y., Ohtsuka, T., Higa, S., Kitajima, I., Demmers,
J., Galjart, N., Houtsmuller, A. B., Grosveld, F., and Akhmanova, A. (2006) CLASPs attach
microtubule plus ends to the cell cortex through a complex with LL5beta. Dev Cell 11, 21-32
30. Salaycik, K. J., Fagerstrom, C. J., Murthy, K., Tulu, U. S., and Wadsworth, P. (2005)
Quantification of microtubule nucleation, growth and dynamics in wound-edge cells. J Cell Sci
118, 4113-4122
FOOTNOTES *This work was supported by grants from DFG to J.B. (Be 1550/6-1) and A.S. (SCHA965/6-1).
The abbreviations used are:
Amer2, APC membrane recruitment 2; APC, adenomatous polyposis coli; EB1, end binding protein 1;
MO, morpholino; PtdIns(4,5)P2, phosphatidylinositol(4,5)bisphosphate; PIP3, phosphatidyl-
inositol(3,4,5)trisphosphate; WTX, Wilms Tumor gene on the X-chromosome.
Conflict of interest The authors declare they have no conflict of interest.
Acknowledgements
We thank M. Brückner for technical and A. Döbler for secreterial assistance. This work was funded by
grants from DFG to J.B. (Be 1550/6-1) and A.S. (SCHA965/6-1).
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FIGURE LEGENDS
Figure 1. Amer2 interacts with EB1 and recruits it to the plasma membrane. (A) Schemes of the human Amer2 protein (18) and the Amer2 bait (amino acids 432-671) used in the
yeast two hybrid screen. A1 and A2 denote APC interacting domains and SKIP and TKIP the EB1 binding
sequence motifs. (B) Scheme of the EB1 protein with microtubule binding domain (MT), linker region (L)
and C-terminal +TIP binding domain (C). EB1 prey clones from the yeast two hybrid screen are aligned
below. (C) EB1-GFP but not GFP co-immunoprecipitates with Flag-Amer2 after transient expression in
HEK293T cells. Western blots were probed with GFP and Flag antibodies. The double band for Amer2
reflects two splice variants (18). Note that relative amounts of these variants vary between different
experiments. Numbers indicate kDa. (D) Endogenous EB1 co-immunoprecipitates with transiently
transfected Flag-Amer2 in HEK293T cells. “-“, indicates transfection of empty Flag vector. Numbers
indicate kDa. (E) Amer2 recruits EB1 to the plasma membrane, whereas Amer1 does not. MCF-7 cells
transiently transfected with Flag-Amer2 or Flag-Amer1 and EB1-GFP were stained with anti-tag
antibodies as indicated in the panels. Boxes in middle panels are magnified in (F). (F) Colocalisation of
Amer2 and EB1 along filamentous structures (arrowheads) and at the plasma membrane (asterisks) in the
boxed area of (E). Transfections and stainings are as in (E). (G) Amer2 recruits endogenous EB1 from
microtubule comets to the plasma membrane in MCF-7 cells transiently transfected with Flag-Amer2 or
empty Flag vector (“-“) and stained using anti-Flag and anti-EB1 antibodies. Arrowheads point to plasma
membrane association of exogenous Flag-Amer2 colocalizing with endogenous EB1. (E-G) scale bar, 10
µm.
Figure 2. Amer2 directly interacts with EB1 via SKIP and TKIP motifs. (A) Protein sequence alignment of human Amer2 and its mouse, rat and frog orthologues. The EB1
binding motifs SKIP and TKIP are conserved in all analyzed species and highlighted by black boxes
(identical residues are indicated by asterisks). Numbers below the sequences indicate the amino acid
residue positions of human Amer2. (B) Mutation of both EB1-binding motifs (IP to NN) in the human
Amer2 bait abolishes interaction with EB1 prey (cf. Fig. 1B) as shown by plate growth and quantitative β-
galactosidase assays in yeast two hybrid experiments. Results of representative experiments are shown.
WT, wild type Amer2 sequence (SKIP/TKIP). (C) Effect of mutating the SKIP and TKIP motifs of
fullsize Amer2 on the EB1 interaction. Co-immunoprecipitation of Flag- Amer2 mutants and EB1-GFP
after transient transfection of HEK293T cells. Immunoprecipitation was performed using Flag sepharose
and Western blots were detected by GFP and Flag antibodies. Numbers indicate kDa. (D) Flag-Amer2-
SKNN/TKNN does not recruit endogenous EB1 to the plasma membrane. Immunofluorescence stainings
of MCF-7 cells transiently transfected with Flag-Amer2 mutants as indicated above the panels. Cells were
stained with anti-Flag and anti-EB1 antibodies. Dashed lines indicate transfected cells. Scale bar, 10 µm.
Figure 3. APC links EB1 to Amer2. (A) Expression of APC promotes co-immunoprecipitation of EB1-GFP with Flag-Amer2 after transient
transfection of HEK293T cells. Flag-Amer1 serves as negative control. Co-immunoprecipitation was
performed using Flag sepharose, Western blots were detected by anti-tag antibodies and anti-APC
antibodies. Numbers indicate kDa. (B) Schematic representation of the Amer2-EB1-APC complex.
Figure 4. Amer2 stabilizes microtubules by interacting with EB1. (A-D) Stabilization of microtubules by Amer2 and EB1. (A) MCF7 cells transiently transfected with Flag-
Amer2 and EB1-GFP (a,b), Flag-Amer2-SKNN/TKNN and EB1-GFP (c,d) and Flag-Amer2 alone (e,f)
were stained for Amer2 and EB1 using anti-tag antibodies, and for acetylated tubulin as indicated in the
panels. Scale bar 10 µm. Arrowheads point to colocalization of acetylated tubulin and EB1-GFP at the
plasma membrane. (B) siRNA mediated knockdown of Amer2 reduces acetylated tubulin levels in
transiently transfected HEK293T (left) and HeLa (right) cells. Cell extracts were probed for Amer2 and
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acetylated tubulin by Western blotting of membrane fractions and whole cell lysates, respectively. Pan-
Cadherin and GAPDH were probed for normalization. Numbers indicate kDa. (C) Knockdown of Amer2
by siRNA in HeLa cells diminishes stabilized microtubules as shown by immunofluorescence staining for
acetylated tubulin; scale bar 20 µm. (D) Knockdown of Amer2, APC and EB1 reduces acetylated tubulin
levels. HEK293T cells transiently transfected with indicated siRNAs were probed for acetylated tubulin,
Amer2, APC, EB1 by either Western blotting (WB) or RT-PCR. α-tubulin and GAPDH were probed for
normalization. Numbers indicate kDa. (E) U2OS cells transfected with indicated plasmids for 1 day were
treated with low doses of nocodazole (0.2µg/ml) for 1 hour and stained for α-tubulin and EB1 (anti-GFP).
Arrowheads point to focal retention of microtubules at areas of EB1 membrane localization.
Magnifications of the boxed regions are shown in the right lower corners. In the merge cell nuclei were
stained with DAPI. Scale bar, 10 µm.
Figure 5. Amer2 is required for cell migration in U2OS cells and in Xenopus embryos. (A) Wound healing assay. Bar chart shows percentage of wound closure by siRNA transfected U2OS cells
12 hours after wounding. Representative immunofluorescence images of α-tubulin stained cells are shown
below. Error bars indicate standard error of the mean. Differences are statistically significant (p<0.05).
Scale bar, 200 µm. (B) Depletion of XAmer2 by XAmer2-MO induces convergent extension defects.
Embryos were scored for anterior extension of the XPAPC expressing paraxial mesoderm as well as
length and width of the negatively stained notochord. Green= normal convergent extension,
orange=moderate convergent extension defects, i.e. broadened and shortened mesoderm tissues,
red=severe gastrulation defects, no convergent extension movements observable. The graph shows the
statistics of four independent experiments with numbers of embryos given below.
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and Juergen BehrensAstrid S. Pfister, Michel V. Hadjihannas, Waldemar Roehrig, Alexandra Schambony
migrationAmer2 interacts with EB1 and APC and controls microtubule stability and cell
published online August 16, 2012J. Biol. Chem.
10.1074/jbc.M112.385393Access the most updated version of this article at doi:
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